Autophagy gene ATG5 knockdown upregulates apoptotic cell death during Candida albicans infection in human vaginal epithelial cells.

PROBLEM: Candida albicans infection induces damage in host cells that results in the activation of cell death pathways such as apoptosis and necrosis. Autophagy acts as a pro-survival mechanism during various infections and has cross talks with apoptosis. The objective of our study was to delineate the effect of autophagy knockdown in human vaginal epithelial cells (VECs) during Candida infection-induced apoptosis. METHOD OF STUDY: We overexpressed wild-type or mutant form of autophagy-related gene 5 (ATG5) in human VECs and determined the levels of autophagy and lysosome marker genes upon C. albicans infection. We analyzed the expression of apoptosis and necrosis markers using confocal microscopy and flow cytometry. RESULTS: Mutant ATG5 overexpressing VECs were unable to form Atg5-Atg12 complex, which is required for functional autophagy pathway. Upon Candida albicans infection, wild-type ATG5 overexpressing cells showed upregulation of autophagy marker genes, ATG5, LC3-I, LC3-II, and LAMP-1. Mutant ATG5 overexpressing cells could not upregulate LC3-II and LAMP-1 expression, indicating a defective autophagy pathway. Confocal microscopy and flow cytometry results revealed that wild-type ATG5 overexpressing VECs showed significantly lower number of apoptotic and necrotic cells as compared to mutant ATG5 overexpressing cells. CONCLUSION: Vaginal epithelial cells with active autophagy were able to survive the damage caused by C. albicans infection, whereas those with defective autophagy succumbed to the infection. This suggests that autophagy plays a critical role in human VECs survival during C. albicans infection.

Transcription factor CCAAT/enhancer-binding protein-ß upregulates microRNA, let-7f-1 in human endocervical cells.

PROBLEM: In endocervical epithelial cells (End1/E6E7), miRNA let-7f plays an important role in the control of innate immunity. The underlying molecular mechanism for let-7f regulation in these cells remains largely unclear. METHODS OF STUDY: let-7f was knocked down in End1/E6E7 cells using siRNA, and differential gene expression was analyzed by microarray. Differentially expressed genes were validated by qPCR and Western blot. Expression of let-7f was studied by qPCR after inhibition of C/EBPß with betulinic acid (BA) and pCMVß plasmid and after overexpression of C/EBPß with pCMVß+ plasmid. ChIP assay was performed to confirm binding of C/EBPß to let-7f promoter. Levels of Lin28A/B were checked by qPCR after similar treatment. RESULTS: let-7f knockdown (KD) affects the expression of many transcription factors (eg, C/EBPß) which are important regulators of immune responses. We observed let-7f-1 promoter to contain 6 C/EBPß binding sites. KD of C/EBPß led to decreased let-7f expression while overexpression of C/EBPß increased its expression. Treatment of End1/E6E7 cells with TLR-3 ligand, poly(I:C) increased binding of C/EBPß at binding sites 3, 5, and 6. Expression of Lin28A/B also changed upon inhibition and overexpression of C/EBPß. Its expression is opposite to that of let-7f in End1/E6E7 cells. CONCLUSION: let-7f-1 is a direct target of transcription factor, C/EBPß in End1/E6E7 cells.

Retinoic acid triggers c-kit gene expression in spermatogonial stem cells through an enhanceosome constituted between transcription factor binding sites for retinoic acid response element (RARE), spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1) and E26 transformation-specific (ETS).

Restricted availability of retinoic acid (RA) in the testicular milieu regulates transcriptional activity of c-kit (KIT, CD117), which aids in the determination of spermatogonial stem-cell differentiation. The effect of RA on c-kit has been reported previously, but its mode of genomic action remains unresolved. We studied the molecular machinery guiding RA responsiveness to the c-kit gene using spermatogonial stem-cell line C18-4 and primary spermatogonial cells. A novel retinoic acid response element (RARE) positioned at -989 nucleotides upstream of the transcription start site (TSS) was identified, providing a binding site for a dimeric RA receptor (i.e. retinoic acid receptor gamma (RARγ) and retinoic X receptor). RA treatment influenced c-kit promoter activity, along with endogenous c-kit expression in C18-4 cells. A comprehensive promoter deletion assay using the pGL3B reporter system characterised the region spanning -271bp and -1011bp upstream of the TSS, which function as minimal promoter and maximal promoter, respectively. In silico analysis predicted that the region -1011 to +58bp comprised the distal enhancer RARE and activators such as spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1), specificity protein 1 (SP1) and four E26 transformation-specific (ETS) tandem binding sites at the proximal region. Gel retardation and chromatin immunoprecipitation (ChIP) assays showed binding for RARγ, PU.1 and SP1 to the predicted consensus binding sequences, whereas GABPα occupied only two out of four ETS binding sites within the c-kit promoter region. We propose that for RA response, an enhanceosome is orchestrated through scaffolding of a CREB-binding protein (CBP)/p300 molecule between RARE and elements in the proximal promoter region, controlling germ-line expression of the c-kit gene. This study outlines the fundamental role played by RARγ, along with other non-RAR transcription factors (PU.1, SP1 and GABPα), in the regulation of c-kit expression in spermatogonial stem cells in response to RA.

Transcriptional regulation of Hb-α and Hb-ß through nuclear factor E2-related factor-2 (Nrf2) activation in human vaginal cells: A novel mechanism of cellular adaptability to oxidative stress.

PROBLEM: Hemoglobin (Hb), a major protein involved in transport of oxygen (O2 ), is expressed by erythroid lineages. Until recently, it was not known whether non-erythroid cells express Hb. The objective was to evaluate the expression and functional significance of Hb-α and Hb-ß in human primary vaginal epithelial cells (hPVECs) and decipher downstream signaling. METHODS OF STUDY: RT-PCR, qRT-PCR, flow cytometry, Western blot, immunofluorescence were used to evaluate the expression of Hb-α, Hb-ß, and nuclear factor E2-related factor-2(Nrf2) after hydrogen peroxide (H2 O2 ) induction. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay were used to determine the binding efficiency of Nrf2 on the Hb-α promoter. RESULTS: Stimulation of hPVECs and human vaginal epithelial cell line, VK2/E6E7 with H2 O2 augmented the expression of Hb-α, Hb-ß, Nrf2, heme oxygenase-1 (HO-1), and reactive oxygen species (ROS). Treatment of these cells with Nrf2 inhibitor, trigonelline (Trig) inhibited Hb-α and Hb-ß expressions. Hb-α and Hb-ß overexpression downregulated H2 O2 -induced ROS. The presence of Nrf2 binding domain was demonstrated within Hb-α promoter. CONCLUSION: The results revealed for the first time that Hb-α and Hb-ß were induced by oxidative stress through the activation of Nrf2. Overexpression of Hb-α and Hb-ß ameliorated H2 O2 -induced oxidative stress, indicating one of the possible mechanism(s) to protect hPVECS from oxidative stress.

Human vaginal epithelial cells augment autophagy marker genes in response to Candida albicans infection.

PROBLEM: Autophagy plays an important role in clearance of intracellular pathogens. However, no information is available on its involvement in vaginal infections such as vulvo-vaginal candidiasis (VVC). VVC is intimately associated with the immune status of the human vaginal epithelial cells (VECs). The objective of our study is to decipher if autophagy process is involved during Candida albicans infection of VECs. METHODS OF STUDY: In this study, C. albicans infection system was established using human VEC line (VK2/E6E7). Infection-induced change in the expression of autophagy markers like LC3 and LAMP-1 were analyzed by RT-PCR, q-PCR, Western blot, immunofluorescence and transmission electron microscopy (TEM) studies were carried out to ascertain the localization of autophagosomes. Multiplex ELISA was carried out to determine the cytokine profiles. RESULTS: Analysis of LC3 and LAMP-1 expression at mRNA and protein levels at different time points revealed up-regulation of these markers 6 hours post C. albicans infection. LC3 and LAMP-1 puncti were observed in infected VECs after 12 hours. TEM studies showed C. albicans entrapped in autophagosomes. Cytokines-TNF-α and IL-1ß were up-regulated in culture supernatants of VECs at 12 hours post-infection. CONCLUSION: The results suggest that C. albicans invasion led to the activation of autophagy as a host defense mechanism of VECs.

Expression of hemoglobin-α and ß subunits in human vaginal epithelial cells and their functional significance.

Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). It consists of Hb-α and Hb-ß subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish in-vitro culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-ß proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-ß proteins (4) to decipher the significance of the Hb-α and Hb-ß expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-ß at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10µg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-κB to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11-7082 treatment (5µM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-ß. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF-κB to Hb-α promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-α and Hb-ß and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-κB regulatory sequences in Hb-α promoter, whose activation is associated with immune response of hPVECs, indicating Hb-α and Hb-ß may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.

HbAHP-25, an In-Silico Designed Peptide, Inhibits HIV-1 Entry by Blocking gp120 Binding to CD4 Receptor.

Human Immunodeficiency Virus (HIV-1) poses a serious threat to the developing world and sexual transmission continues to be the major source of new infections. Therefore, the development of molecules, which prevent new HIV-1 infections, is highly warranted. In the present study, a panel of human hemoglobin (Hb)-α subunit derived peptides and their analogues, with an ability to bind gp120, were designed in-silico and their anti-HIV-1 activity was evaluated. Of these peptides, HbAHP-25, an analogue of Hb-α derived peptide, demonstrated significant anti-HIV-1 activity. HbAHP-25 was found to be active against CCR5-tropic HIV-1 strains (ADA5 and BaL) and CXCR4-tropic HIV-1 strains (IIIB and NL4-3). Surface plasmon resonance (SPR) and ELISA revealed direct interaction between HbAHP-25 and HIV-1 envelope protein, gp120. The peptide prevented binding of CD4 to gp120 and blocked subsequent steps leading to entry and/or fusion or both. Anti-HIV activity of HbAHP-25 appeared to be specific as it failed to inhibit the entry of HIV-1 pseudotyped virus (HIV-1 VSV). Further, HbAHP-25 was found to be non-cytotoxic to TZM-bl cells, VK2/E6E7 cells, CEM-GFP cells and PBMCs, even at higher concentrations. Moreover, HbAHP-25 retained its anti-HIV activity in presence of seminal plasma and vaginal fluid. In brief, the study identified HbAHP-25, a novel anti-HIV peptide, which directly interacts with gp120 and thus has a potential to inhibit early stages of HIV-1 infection.

MicroRNA let-7f: a novel regulator of innate immune response in human endocervical cells.

PROBLEM: Endocervical epithelial cells express pattern recognition receptors (PRRs) that aid in innate immune responses. Mechanisms regulating signaling of PRRs are poorly understood. METHODS OF STUDY: Endocervical cells (End1/E6E7) were treated with ligands of TLR9 and RIG-I once or after pre-stimulation with same ligand. Cytokine responses were determined by ELISA. Differential gene expression was analyzed by microarray. Differentially expressed genes were validated by qPCR /Western blot. Role of let-7f was studied by inhibition and over-expression studies using commercial inhibitors and let-7f encoding plasmids, respectively. RESULTS: Single stimulation of cells with TLR9 ligand, but not RIG-I ligand, induced tolerance to subsequent challenge to the same ligand. Stimulation with TLR9 decreased let-7f and increased its target Blimp-1. Conversely, RIG-I stimulation increased let-7f and decreased Blimp-1 expression. Inhibition and over-expression revealed let-7f is involved in induction of immune tolerance. CONCLUSION: We identify let-7f as a novel regulator of PRR signaling in endocervical cells.

TLR9 and RIG-I signaling in human endocervical epithelial cells modulates inflammatory responses of macrophages and dendritic cells in vitro.

The innate immune system has evolved to recognize invading pathogens through pattern recognition receptors (PRRs).Among PRRs, Toll like receptors (TLRs 3, 7/8,9) and RIG-I like receptors (RLRs) have been shown to recognize viral components. Mucosal immune responses to viral infections require coordinated actions from epithelial as well as immune cells. In this respect, endocervical epithelial cells (EEC's) play an important role in initiating innate immune responses via PRRs. It is unknown whether EEC's can alter immune responses of macrophages and dendritic cells (DC's) like its counterparts in intestinal and respiratory systems. In this study, we show that endocervical epithelial cells (End1/E6E7) express two key receptors, TLR9 and RIG-I involved in anti-viral immunity. Stimulation of End1/E6E7 cells lead to the activation of NF-κB and increased secretion of pro-inflammatory cytokines, IL-6 and IL-8. Polarized End1/E6E7 cells responded to apical stimulation with ligands of TLR9 and RIG-I, CpG-ODN and Poly(I:C)LL respectively, without compromising End1/E6E7 cell integrity. At steady state, spent medium from End1/E6E7 cells significantly reduced secretion of pro-inflammatory cytokines from LPS treated human primary monocyte derived macrophages (MDMs) and DC:T cell co-cultures. Spent medium from End1/E6E7 cells stimulated with ligands of TLR9/RIG-I restored secretion of pro-inflammatory cytokines as well as enhanced phagocytosis and chemotaxis of monocytic U937 cells. Spent medium from CpG-ODN and Poly(I:C)LL stimulated End1/E6E7 cells showed significant increased secretion of IL-12p70 from DC:T cell co-cultures. The anti-inflammatory effect of spent media of End1/E6E7 cell was observed to be TGF-ß dependent. In summary, the results of our study indicate that EEC's play an indispensable role in modulating anti-viral immune responses at the female lower genital tract.

Identification, cloning, characterization and recombinant expression of an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab, Scylla serrata.

Anti-lipopolysaccharide factors (ALF) are a group of small basic proteins which are released into the hemolymph as a result of rapid degranulation of hemocytes in response to bacterial lipopolysaccharide (LPS). In the present study, using a combined approach of degenerate and RACE PCR, the gene coding for Scylla serrata anti-lipopolysaccharide factor (SsALF) was cloned and characterized. The full-length SsALF cDNA sequence consists of 607 bp and encodes a polypeptide of 97 amino acids, constituting a molecular mass of 11172 Da with an estimated pI of 10.01. The SsALF protein showed upto 92% similarity with ALF from Scylla paramamosain and about 33-53% amino acid sequence identity with other known ALF sequences. SsALF protein sequence demonstrated the presence of two highly conserved cysteine residues and putative LPS binding domain. An in vivo expression study showed that SsALF mRNA was expressed predominantly in hemocytes, heart and muscle of healthy mud crabs. The recombinant form of SsALF protein (rSsALF) was expressed with a Histag, in Escherichia coli, using the pTriEx-4 Ek/LIC vector. The purified rSsALF protein demonstrated antimicrobial activity against both Gram-positive and Gram-negative bacteria. The recombinant protein was able to significantly neutralize LPS-induced expression on SsALF in vivo as demonstrated by real-time PCR. rSsALF was able to permeabilize artificial phospholipid membranes as demonstrated by calcein enclosed liposome model. These studies strongly suggest that SsALF is one among the important antimicrobial factors produced in the crab during a microbial invasion.

Purification and characterization of antibacterial proteins from granular hemocytes of Indian mud crab, Scylla serrata.

Marine invertebrates depend upon antimicrobial peptides (AMPs) as a major component of innate immunity, as they are rapidly synthesized and diffuse upon pathogen invasion. In this study, we report the identification and characterization of a 11 kDa antimicrobial protein, which we name SSAP (for Scylla serrata antimicrobial protein), from granular hemocytes of the mangrove crab S. serrata. The protein is highly similar to scygonadin, a male-specific AMP isolated from the ejaculatory duct of S. serrata. SSAP was isolated using various chromatographic techniques, viz. ion-exchange, ultra filtration and RP-HPLC, and demonstrated antibacterial activity against Gram positive and Gram negative bacteria. Full length mRNA encoding SSAP was amplified using a combination of RT-PCR and RACE. The nucleotide sequence revealed a full-length ORF of 381 bp coding for a preprotein of 126 amino acids comprising a signal peptide of 24 amino acids and a mature protein of 102 amino acids with a predicted mass of 11435 Da and pI of 5.70. Unlike scygonadin, SSAP is expressed in several tissues of both male and female crabs, as evidenced by RT-PCR, Northern and Western blot analyses. The study suggests that SSAP might be an isoform or a variant of scygonadin and might play an important role in regulating the immunity of the crab upon microbial infection.